Protein Phosphorylation And Regulation of Cytoskeleton in The Nervous System: In our previous studies on the squid giant fiber system, we examined the factors regulating compartment-specific patterns of cytoskeletal protein phosphorylation (primarily neurofilaments), in an effort to establish a normal baseline of information for further studies of neurodegeneration. It is striking that neurofilament proteins contain over 100 phosphate acceptor sites in a series of proline-directed serine / threonine (S/T-P) repeat motifs, all of which are phosphorylated exclusively in the axon by proline directed S/T- kinases such as cdk5. A similar pattern is also found in mammalian neurofilament proteins. In many neurodegenerative disorders that lead to dementia, or to muscle degeneration such as in ALS, the pathology responsible for neuronal loss is marked by accumulations in cell bodies of filamentous tangles and deposits containing hyperphosphorylated cytoskeletal proteins. These are assumed to arise as a consequence of deregulation of a normal pattern of topographic phosphorylation, i.e., an aberrant cytoskeletal protein phosphorylation in the cell body compartment. Deregulation has also been shown to involve the hyperactivity of cdk5, normally tightly regulated during development of the nervous system, where it modulates neuronal differentiation, migration, synaptic function and survival. Our laboratory has taken a two-pronged approach on the problem by concentrating on the cytoskeletal proteins, their synthesis, processing and phosphorylation in different compartments, while at the same time we have studied the multifunctional role of cdk5 in other aspects of nervous system function. Our past studies have shown that in addition to its role in phosphorylating cytoskeletal proteins in normal and stressed neurons, as well as synaptic proteins, the diverse functions of cdk5 stem from its cross talk interactions with several signaling pathways implicated in neuronal function and survival. For example we have shown that cdk5 downregulates the JNK apoptotic pathway and up regulates the PI3K/AKT survival pathway. More recently we have shown that cdk5 modulates another key protein in signal transduction, it phosphorylates and down regulates Ras GRF1, a protein responsible for the activation of Ras and RacGTPases, upstream kinases in several signaling pathways. Here,its effect was most pronounced on nuclear organization, since overexpression of RasGRF1 or inhibition of cdk5 resulted in condensation and fragmentation of neuronal nuclei. Though cdk5 plays a key role in post-mitotic cortical neuron migration during development, its uncertain whether early determination and differentiation of neurons requires cdk5 activity. To explore this issue we chose the zebra fish in which early development is experimentally accessible, more so than in the mammal. We were able to show that,indeed,cdk5 activity is expressed early, that knocking down cdk5 by microinjection of cdk5 SiRNA, the number of Rohon-Beard (RB) cells (the earliest primary sensory neurons along the dorsal spinal cord) were significantly reduced and restored only after injection of cdk5 mRNA. Since RB cells normally die after two days, it was shown that injection of cdk5 mRNA at the 1-2 cell stage prevented RB cell apoptosis suggesting that cdk5 may promote both RB cell differentiation as well as survival. In a recent study, in collaboration with Dr. Kulkarni?s Laboratory (NIDCR, NIH), we have further examined cdk5's role during synaptic function to determine whether cdk5 was involved in pain nocioceptive signaling in adult mice. It was demonstrated that cdk5/p35 were expressed in nocioceptive neurons, that a peripheral inflammatory response increased calpain activity and the cleavage of p35 to p25, a more active and stable regulator of cdk5, thereby increasing kinase activity and pain sensitivity. Sensitivity to pain depended on cdk5 activity since p35 knockout mice with significantly reduced activity were less sensitive to thermal stimuli. The data suggest that cdk5 may modulate the expression of the pain receptor by phosphorylation, a hypothesis that is currently under investigation.